Molecular Human Reproduction, Vol. 5, No. 6, 581-586,
June 1999
© 1999 European Society of Human Reproduction and Embryology
Leukaemia inhibitory factor gene mutations in infertile women
1 Department of Neurology (Clinical Research Group), and 2 Department of Gynaecology and Obstetrics, Julius-Maximilians-University of Würzburg, Josef Schneider Straße 11, 97080 Würzburg, Germany
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Abstract |
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The glycoprotein leukaemia inhibitory factor (LIF) is produced by the endometrium and is involved in the control of implantation. In women with unexplained infertility reduced uterine concentrations of LIF have been reported. Studies with mice lacking a functional LIF gene have shown that the LIF protein is essential for implantation of the embryo. We have developed a method for screening of gene mutations in the coding region and critical regulatory regions of the LIF gene. Thus we could screen nulligravid infertile women (n = 74), fertile controls (n = 75) and as a second unrelated control group, neurological patients (n = 131) for LIF gene mutations. In infertile women, three heterozygous point mutations have been identified: one in close proximity to the start codon of exon 1 and two mutations in exon 3. These correspond to regions of the LIF protein which are thought to be highly important for interaction with the LIF receptor and thus lead to reduced biological activity of the LIF protein. Only one point mutation/polymorphism in the non-coding region between exon 2 and 3 was found in the control groups. Our results suggest that heterozygosity for a LIF gene mutation could give rise to decreased availability or biological activity of LIF in the uterus and cause implantation failure. Thus the mutations identified in our study could be responsible for infertility in a subgroup of nulligravid women.
infertility/leukaemia inhibitory factor (LIF)/mutations
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Introduction |
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Implantation failure is a major reason of infertility in otherwise healthy women. Despite new treatment strategies, particularly in the area of in-vitro fertilization (IVF), only 11–17% of embryos grown in vitro achieve successful implantation (Imoedemhe et al., 1995
; van der Elst et al., 1996). Genetic reasons are suspected, but the underlying gene alterations are not understood.
Leukaemia inhibitory factor (LIF) is a glycoprotein with a variety of functions in different organ systems. Originally, it was identified as a differentiation factor for haematopoietic cells, but has since been shown to support diverse physiological functions. Among them are the inhibition of differentiation in embryonic stem cells, the stimulation of differentiation and survival effects in neuronal cells, and the involvement in complex defence reaction such as acute phase induction in liver cells and other cell types of the immune system (Gearing et al., 1992; Metcalf, 1992; Escary et al., 1993; Robinson et al., 1994).
Mice in which the LIF gene was inactivated developed normally and did not show any severe phenotypic alterations during post-natal life with one exception: female mice with homozygous LIF gene inactivation could not become pregnant (Stewart et al., 1992). Moreover, treatment of mice leading to increased LIF mRNA values in the uterus promotes embryo implantation (Takabatake et al., 1997). This pointed to an important function of the LIF protein in reproduction. LIF protein is normally produced by the endometrium, and several subsequent studies have shown that it is involved in the control of implantation also in human (Dunglinson et al., 1996; Nachtigall et al., 1996).
In order to investigate whether reduced availability of LIF and subsequent infertility could be caused by mutations within the coding regions of the human LIF gene, we have developed techniques for amplification and analysis of all three coding exons of the human LIF gene. We undertook this study to investigate the frequency of functional mutations within coding regions of the LIF gene in a population of women suffering from infertility from various causes. While women with homozygous defects of the LIF gene should be unable to reproduce at all, those with heterozygous mutations that interfere with translation are expected to suffer from reduced bioavailability and/or structural alterations of the LIF gene product. It can be assumed that decreased availability of the LIF protein in the uterus may constitute a relevant cause for the infertility of these patients.
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Materials and methods |
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Patients and controls
We have screened 74 non-selected nulligravid infertile women (median age 31.8 years, range 22–41 years) and 75 fertile controls with at least one live birth (controls I, median age 31.1 years, range 18–50 years) for LIF gene mutations. Patients consulted the Department of Gynaecology and Obstetrics, University of Würzburg, for infertility of >2 years duration from various causes: such as endometriosis (n = 8), polycystic ovarian or tubal disease (n = 25), ovulatory disorders or obesity (n = 13), male subfertility (n = 18), or infertility `sine causa' (n = 10).
In order to investigate the frequency of LIF mutations in a population not selected for obstetric criteria, 131 neurological patients (muscular dystrophy, n = 72, median age 16.1 years, range 1–61 years, all males; ischaemic stroke, n = 59, median age 70.3 years, range 37–88 years, 24 females/35 males) were analysed as a second control group (controls II). The study was approved by the local ethics committee, and all patients gave their signed consent.
Preparation of genomic DNA from human blood samples
Genomic DNA was extracted from EDTA blood samples using a standard method (Miller et al., 1988). EDTA blood (20 ml) was diluted with 30 ml ice-cooled lysis buffer (NH4Cl 155 mM, KHCO3 10 mM, Na2EDTA 0.1 mM; pH 7.4) and incubated for 15 min on ice. After centrifugation (10 min., 1500 rpm, 4°C) the pellet was resolved in 5 ml SE-buffer (NaCl 75 mM, Na2EDTA 25 mM; pH 8.0), 250 µl sodium dodecyl sulphate (SDS; 20%) and 50 µl Pronase E (10 mg/ml). The solution was incubated overnight. After proteolysis, 5 ml SE-buffer and 3 ml of saturated sodium chloride were added. After centrifugation (20 min, 3000 rpm, 25°C) the DNA containing supernatant was removed, and DNA was precipitated by adding 20 ml ethanol. DNA was resolved with 500 µl TE-buffer (Tris–HCl 10 mM, Na2EDTA 1 mM; pH 8.0). The purity of the extracted DNA (usually 150–300 µg/ml) was tested photometrically, the ration of the optical density (OD) at 260/280 nm was 1.7–2.0.
Development and characterization of PCR conditions for amplification of the three coding exons of the human LIF gene
Polymerase chain reaction (PCR) was performed using five primer sets flanking the three exons and parts of the introns of the LIF gene (Stahl et al., 1990). For exon 1, primers 5'-ACT GCC GGC ATC TGA GGT TTC CTC-3' and 5'-GCT GCC AAG CGC CCC AAG TTG CCG-3' were used. For exon 2, set 5'-GCC ACC CTT TCC TGC CTT TCT AC-3' and 5'-TCC CTG CCA TCT CCT GTC AGT ATC-3' was applied. For exon 3, three sets of primers were chosen for amplification of 5' and 3' parts of this exon: set a: 5'-ACA ATT CCA GAT GCT TAC AGG G-3' and 5'-GCC AAG GTA CAC GAC TAT GC-3'; set b: 5'-CCC AAC AAC CTG GAC AAG CTA TG-3' and 5'-CCG TAG GTC ACG TCC ACA TG-3'; set c: 5'-CCT CCA CAG CAA GCT CAA CG-3' and 5'-CGG TTC ACA GCA CAC TTC AAG-3'. The reaction (volume 50 µl) was performed with standardized conditions: 1x PCR buffer (Perkin Elmer, Norwalk CT, USA), 0.2 mM dNTP, 0.25 U AmpliTaq
DNA Polymerase (Perkin Elmer), 20 pmol sense primer, 20 pmol antisense primer. PCR amplification was done using a Perkin Elmer GeneAmp 2400 thermal cycler. Amplification was carried out as follows: 5 min at 94°C initial denaturation; 30 s at 94°C denaturation, 30 s at 65°C (except exon 3, set b at 60°C) annealing, 60 s at 72°C extension, entrained for 34 cycles; 10 min at 72°C final extension. The expected product sizes were 143, 268, 284, 284, 243 bp respectively. In order to avoid errors, all PCRs were repeated at least twice for patients with mutations in the LIF gene.
Single strand conformation polymorphism (SSCP) analysis for investigation of gene mutations within the human LIF gene
PCR product (8 µl) was mixed with 16 µl formamide 95% (containing xylencyanol and bromophenol as marker) and heated to 95°C for 5 min. After cooling on ice for 5 min, 6 µl PCR product/formamide mixture were loaded on a rehydrated native 10% polyacrylamide gel (CleanGel DNA-HP 10% 36S, ETC Corporation, Kirchentellinsfurt, Germany). For rehydration of the gel and for the electrode wicks DNA Disc buffer system (ETC Corporation) was used.
The gel was run in a Multiphor II electrophoresis chamber (Amersham Pharmacia Biotech, Braunschweig, Germany) under the following conditions:
Exon 1: 8°C; 110 V, 16 mA, 6 W for 20 min.; 600 V, 42 mA, 16 W for 85 min.
Exon 2: 8°C; 110 V, 16 mA, 6 W for 20 min.; 600 V, 42 mA, 16 W for 135 min.
Exon 3 set a: 8°C; 110 V, 16 mA, 6 W for 20 min.; 600 V, 42 mA, 16 W for 135 min.
Exon 3 set b: 12°C; 110 V, 16 mA, 6 W for 20 min.; 600 V, 42 mA, 16 W for 120 min.
Exon 3 set c: 12°C; 110 V, 16 mA, 6 W for 20 min.; 600 V, 42 mA, 16 W for 120 min.
Band patterns could easily be detected using a standard silver impregnation method (Bassam et al., 1991) according to the manufacturer's protocol (ETC Corporation) (Figure 1).
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DNA sequencing as a control for the polymorphisms identified by SSCP
We sequenced PCR products which showed abnormal bands in the SSCP analysis using a Perkin Elmer 373 A DNA sequencer (Perkin Elmer). For fluorescent sequencing we performed a second PCR using AmpliTaq
DNA Polymerase (Perkin Elmer) and a Dye Terminator Kit (Perkin Elmer) according to the manufacturer's protocol. For automated, fluorescent, laser-detected DNA sequencing we used a non-native 5.5% polyacrylamide gel. In order to avoid artefacts caused by PCR errors, all sequences were obtained from at least two independently amplified DNA products for patients with mutations in the LIF gene. In those patients also the antisense PCR product was sequenced to confirm the mutation.
DNA databases
Obtained sequences were compared with the published LIF DNA sequence (GenBank). The number of nucleotides in this paper refers to the LIF DNA sequence deposited in GenBank, Accession number M63420.
Statistical analysis
The groups were compared for statistically significant differences using the 
2 test.
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Results |
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Development of a PCR-based method to screen for mutations in the human LIF gene
Various sets of flanking primers were investigated in order to identify optimal conditions for amplification of the DNA coding for the exons of the human LIF gene. Exon 1 could be amplified by one primer pair covering 50 nucleotides from the 5' non-coding region, the entire coding region of 19 nucleotides of the signal peptide and 74 nucleotides from the 3' non-coding region (nucleotides 671–813). A similar technique could be established for the amplification of exon 2 using a primer set for amplifying a region spanning 50 nucleotides from the 5' non-coding region and 39 nucleotides from the 3' non-coding region (nucleotides 2421–2688). Exon 3 is the largest exon, and attempts to amplify this exon in its entirety did not prove useful for subsequent SSCP analysis. Therefore, exon 3 was amplified by the use of three sets of primer pairs (sets a, b, c), covering the 5' region including 143 nucleotides of the 5' non-coding region to the 3' region including 32 nucleotides of the 3' non-coding region (nucleotides 3200–3782). The first primer set `a' covers 143 nucleotides of the 5' non-coding region and 141 nucleotides of the coding sequence (nucleotides 3200–3483). The second primer set `b' covers the region corresponding to amino acids 53–147 of the protein (nucleotides 3367–3650), and the third primer set `c' flanks amino acids 111–180 of the protein and 32 nucleotides of the 3' non-coding region (nucleotides 3540–3782). The resulting amplification products showed the following lengths: exon 1: 143 nucleotides, exon 2: 268 nucleotides, exon 3a: 284 nucleotides, 3b: 284 nucleotides, 3c: 243 nucleotides. Thus an ideal length of <300 nucleotides for subsequent SSCP analysis could be obtained.
SSCP analysis and subsequent DNA sequencing revealed point mutations in the human LIF gene
In infertile women (n = 74), we identified three point mutations; one in the non-coding region of exon 1 and two mutations in the coding region of exon 3 (Table I). The three women with mutations in the LIF gene had attempted to become pregnant for 4–7 years, and none showed any other precondition rendering them totally infertile (e.g. bilateral occlusion, or male azoospermia). All three women had regular ovulatory cycles. Two were suffering from pelvic endometriosis and were treated with IVF (one and four cycles respectively). The other woman was diagnosed as having pelvic adhesions with patent tubes and was treated by ovulation induction in combination with intrauterine insemination (three cycles) for concomitant mild-to-moderate male subfertility.
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The mutation in exon 1 was a C
A transition at position 715 of the human LIF gene, 6 bp 5' from the start codon. The mutations in exon 3 were two GA transitions at positions 3400 and 3424 of the human LIF gene leading to an exchange of valine to methionine at position 64 of the mature LIF protein, and an exchange of alanine to threonine at position 72 respectively. These positions correspond to regions of the LIF protein (AB loop) which are thought to be highly important for interaction with the LIF-receptor (Robinson et al., 1994).
Only one individual from both control groups (n = 206) showed a LIF gene polymorphism, which consisted in a CT transition at position 3235 within the intron between exon 2 and 3, and therefore is not expected to alter protein conformation or expression levels. All point mutations observed in this study were heterozygous (Figures 2 and 3).
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Discussion |
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LIF is a multifunctional glycoprotein which has an important role in reproduction (Senturk and Arici, 1998
). LIF is expressed in the human endometrium in a menstrual cycle-dependent manner. Maximal expression patterns were observed on days 19–25 of the menstrual cycle coinciding with time of blastocyst implantation (Arici et al., 1995). LIF also enhances blastocyst formation of human embryos and modulates trophoblast differentiation in vitro, and thus induces conditions necessary for implantation (Dunglinson et al., 1996; Nachtigall et al., 1996). Marked cyclical changes of LIF immunoreactivity in the human endometrial epithelium suggest a paracrine/autocrine role for LIF in endometrial function (Vogiagis et al., 1996), which is supported by investigations of the distribution of the LIF-receptor complex consisting of LIF receptor ß and gp130 (Yang et al. 1995; Cullinan et al., 1996; Owczarek et al., 1996). LIF receptor ß expression is restricted to the luminal epithelium during the proliferative and secretory phase of the cycle. The associated signal transducing component of the LIF-receptor, gp 130, is also expressed in both the luminal and glandular epithelium throughout the cycle (Cullinan et al., 1996).
The amount of LIF in flushings obtained from women with unexplained infertility was shown to be significantly lower than those from fertile women on day LH+10 (Laird et al., 1997). Furthermore, Hambartsoumian (1998) showed differences of LIF secretion in endometrial explant cultures between fertile and infertile women. In fertile women, the endometrial LIF secretion was 2.2 higher in the secretory phase than in the proliferative phase, whereas infertile women did not exhibit such an elevation of LIF production in the secretory phase. LIF production in fertile women on days 18–21 of the menstrual cycle was 3.5 higher than in infertile women with multiple failures of implantation, and 2.2 times higher than in infertile women without multiple failures of implantation.
These alterations point to the presence of polymorphisms and gene mutations on the DNA level, which could contribute to reduced production and biological activity of the LIF protein in the endometrium. In order to test this possibility, we have developed a technique for rapid screening of LIF gene mutations. This method revealed biologically relevant mutations in infertile women. The gene mutations observed were heterozygous, and in none of the cases was a homozygous gene mutation detected.
We assume that the heterozygous LIF gene mutations found in our study could be responsible for a decreased LIF protein production which was described by Laird et al. (1997) and Hambartsoumian (1998). One mutation observed in our screen (Table I
) was located in the regulatory regions 5' from the start codon of the LIF gene. Two other mutations were identified in positions that correspond to regions of the LIF protein (AB loop), which are thought to be highly important for the interaction with the LIF receptor (Robinson et al., 1994). Only one individual from both control groups (n = 206) showed a LIF gene polymorphism in the non-coding intron region between exons 2 and 3, which is, therefore, not expected to alter protein conformation or expression levels. Thus, the frequency of functionally relevant mutations of the LIF gene in infertile women is significantly enhanced in comparison with controls (P < 0.05, 2 test).
The presence of one intact allele suggests that the amount of LIF protein produced by the endometrium in these patients should be reduced by 
50%. Unfortunately, we were not able to determine LIF protein values by uterine flushings or endometrial biopsies in our patient group. A recent study has shown that LIF protein concentrations in endometrial biopsies of infertile women show a high degree of variation (Hambartsoumian, 1998). Therefore, we suspect that reduction of the LIF protein concentration in infertile women could at least in part be caused by genetic defects of the LIF gene. Possibly such genetic predispositions could work together with other reasons for infertility in patients.
Studies with mice lacking a functional LIF gene have shown that embryos fail to implant, indicating that maternal LIF is essential for successful pregnancy. However, implantation failure was partially compensated by application of LIF in the peritoneal cavity of these LIF-deficient mice (Stewart et al., 1992). Based on these studies, LIF substitution in infertile women with LIF gene mutations might serve as a therapeutic option in this subgroup of infertile women, and the techniques for screening such gene mutations could help to identify this subgroup of patients.
We conclude that heterozygosity for a LIF gene mutation leading either to decreased availability or decreased specific biological activity of the LIF protein respectively, may be a cause of either failure or decreased efficacy of implantation, and thus be responsible for infertility in a subgroup of nulligravid women. Since our patient group of infertile patients also consisted of infertile women with obvious reasons for infertility (endometriosis, polycystic ovarian or tubal disease, ovulatory disease or obesity, male subfertility) further analysis of infertile women with otherwise unexplained infertility has to show whether an increased frequency of mutations in the LIF gene can be observed in subgroups of infertile women.
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3 To whom correspondence should be addressed
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References |
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Arici, A., Engin, O., Attar, E. et al. (1995) Modulation of leukaemia inhibitory factor gene expression and protein biosynthesis in human endometrium. J. Clin. Endocrinol. Metab., 80, 1908–1915.[Abstract]
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Submitted on October 27, 1998; accepted on February 23, 1999.
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